Dual role of magnetic nanoparticles as intracellular hotspots and

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Biological and Medical Applications of Materials and Interfaces

Dual role of magnetic nanoparticles as intracellular hotspots and extracellular matrix disruptors triggered by magnetic hyperthermia in 3D cell culture models Lilianne L. Beola, Laura Asín, Raluca Maria M Fratila, Vanessa Herrero, Jesús M. De La Fuente, Valeria Grazu, and Lucía Gutiérrez ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18270 • Publication Date (Web): 27 Nov 2018 Downloaded from http://pubs.acs.org on November 29, 2018

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ACS Applied Materials & Interfaces

Dual Role of Magnetic Nanoparticles as Intracellular Hotspots and Extracellular Matrix Disruptors Triggered by Magnetic Hyperthermia in 3D Cell Culture Models Lilianne Beola†,#, Laura Asín,†,‡, #,* Raluca M. Fratila†,‡, Vanessa Herrero†, Jesús M. de la Fuente†,‡, Valeria Grazú†,‡ and Lucía Gutiérrez†,‡,§*, †Instituto

de Ciencia de Materiales de Aragón (ICMA), CSIC/Universidad de Zaragoza, C/ Pedro Cerbuna 12,

50009, Zaragoza, Spain. ‡Centro

de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN),

50018, Spain. §

Department of Analytical Chemistry, Instituto Universitario de Nanociencia de Aragón (INA), Universidad de

Zaragoza, Edificio I+D, Mariano Esquillor Gómez, 50018, Zaragoza, Spain # Shared first author *L.A.: [email protected]; L.G.: [email protected]

Keywords: iron oxides, magnetic nanoparticles, hyperthermia, 3D cell culture, macrophages, cell death, collagen. Abstract Magnetic hyperthermia is a promising therapy for the localized treatment of cancer based on the exposure of magnetic nanoparticles to an external alternating magnetic field. In order to evaluate some of the mechanisms involved in the cellular damage caused by this treatment, two different 3D cell culture models were prepared using collagen, which is the most abundant protein of the extracellular matrix. The same amount of nanoparticles was added to cells either before or after their incorporation to the 3D structure. Therefore, in one model, particles were located only inside cells (In model), while the other one had particles both inside and outside cells (In&Out model). In the In&Out model, the hyperthermia treatment facilitated the migration of the particles from the outer areas of the 3D structure to the inner parts, achieving a faster homogeneous distribution throughout the whole structure and allowing the particles to gain access to the inner cells. The cell death mechanism activated by the magnetic hyperthermia treatment was different in both models. Necrosis was observed in the In model while apoptosis in the In&Out model 24 hours after the hyperthermia application. This was clearly correlated with the amount of nanoparticles located inside the cells. Thus, the combination of both 3D models allowed us to

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demonstrate two different roles of the magnetic particles during the hyperthermia treatment: i) The modulation of the cell death mechanism depending on the amount of intracellular particles, and ii) The disruption of the collagen matrix caused by the extracellular nanoparticles.

Introduction Iron oxide magnetic nanoparticles (MNPs) are one of the most promising systems within the field of biomedicine and biotechnology.1-3 Their physico-chemical characteristics as well as their magnetic properties, provide unique opportunities for the development of a wide range of applications such as biosensors,4-5 controlled drug delivery systems,6-7 contrast agents for magnetic resonance imaging,8-9 or cancer therapies such as magnetic hyperthermia.10 Magnetic hyperthermia (MH) has been particularly studied in recent years for the treatment of malignant tumours.11-13 It is widely known that cancer cells are more sensitive to heat than normal cells.14 MNPs act as localized heating sources in the region where they are situated when submitted to an alternating magnetic field (AMF).15 The local temperature increase in the tumour area induces cell death or alters the growth and differentiation of the cancer cells.16 At the same time, MH has been used to promote synergistic effects when combined with other conventional treatments. In particular, it has been shown that MH treatment makes the cells more sensitive to radiation or to the action of certain chemotherapeutic drugs.17-19 A possible explanation of this synergistic effect between heat and chemotherapy was given by Krawczyk et al. who demonstrated that mild hyperthermia conditions provoked the degradation of BRCA2, a protein involved in the homologous recombination, which is a DNA repair mechanism by which the double strands breaks are repaired. If the cells are deficient in this DNA repair mechanism they become more sensitive to drugs that induce DNA damage.20 The cell death mechanisms activated by the MH treatment and the relevant parameters that control the triggering of each mechanism are not completely known yet. The classical conception of two cell death routes, apoptosis and necrosis, which take place independently, has been already refuted. Besides, some classical postulates about these two cell death mechanisms have been progressively abandoned and replaced for new ideas. It is known that necrosis can take place in a regulated way, like apoptosis, and that the apoptotic cells can sometimes be recognized by the immune system and trigger an adaptive immune response, like necrosis. Nevertheless, it is crucial to be able to control the activation of either necrosis 2


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or apoptosis mechanisms and to try to control the pro-inflammatory immune response that could be sometimes negative for tumour treatments, as it involves non-desirable processes such as the tumour invasion or metastasis. 21-23 Currently, most of the in vitro studies that analyse the efficacy of the hyperthermia treatment are being performed in monolayer cell cultures.24-26 However, this traditional cell culture method, in two dimensions (2D), has essential limitations in terms of inter-cellular communication, cell microenvironment and cell spatial behaviour.27 Therefore, these 2D cell culture models cannot replicate the morphology and biochemical properties the cells have in the living organisms. They also lack the complex scenario that the MNPs face to reach the deeper areas of the tumours. One of the alternatives to solve these problems is the use of three dimensions (3D) cell culture models, such as spheroids, liquid spheres,28-29 or 3D scaffolds.3031

3D cell culture models are simple structures that mimic more accurately the tumour

composition and structure32 providing a more realistic environment to evaluate the cellular response to a treatment 33. In addition, 3D cell culture models allow the production of a high number of identical replicas and provide a good alternative to perform preliminary tests before in vivo experimentation allowing the reduction of the number of animals needed. Until know, the number of studies using 3D cell culture models to evaluate the effect of magnetic hyperthermia is still scarce.34 These previous studies have mainly used spheroids as 3D cell cultures, a model that lacks the presence of extracellular matrix. In this work, we have developed two 3D cell culture models based on the use of a collagen matrix to evaluate the efficacy of magnetic hyperthermia treatment in a murine macrophage/monocyte cell line (RAW 264.7). Collagen was selected because besides being one of the major components of the extracellular matrix (ECM), it also plays a crucial role in the tumour ECM.35 In particular, tumours with well-organized and highly interconnected collagen fibres display lower penetration of high-molecular-weight chemotherapeutic agents than those with disordered and loose collagen networks.36

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Macrophages were chosen as

model cells due to their known capacity of easily incorporating iron oxide nanoparticles.38 This fact allowed working with a wide range of concentrations of nanoparticles inside the cells, which facilitated the study of the correlation between the amount of internalized particles and the cell death mechanism activated. The main difference between the two models is the moment in which MNPs are in contact with the cells along the 3D cell culture preparation protocol. In one model (In Model), cells in suspension are incubated with the particles and after washing away the MNPs that have not been internalized, the cells are transferred to form the 3D matrix, thus resulting in a homogeneous internalization of the 3



MNPs amongst all the cells and also an exclusive intracellular location of the MNPs. The other model is based on the administration of the MNPs to the cells once they are already forming part of the 3D matrix resulting in a model where particles need time to penetrate and fill up the whole 3D matrix to gain access to inner cells. As a result, this model allows obtaining a different MNP distribution than the former approach, having particles located both inside and outside the cells (In&Out Model) (Fig. 1). The combination of both models allows disentangling the effect that MNPs have on the extracellular matrix and the treatment efficiency. In this work, we have used confocal microscopy and flow cytometry to study the MNP uptake, with and without exposure to an AMF. We have studied the evolution of the MNP location within the collagen matrix after the AMF exposure. We have also evaluated the cell death mechanisms triggered by the treatment at different times post-treatment (0, 24 and 48 h). Cell cycle and the cell viability studies allowed to discriminate the cell death mechanisms observed 24 h after MH treatment which was different on each 3D model: necrosis for the In Mode and apoptosis in the case of the In&Out Model. Besides, we have analysed the amount of MNPs internalized in each 3D model in order to establish a relationship between their concentration and the cell death mechanism observed after the treatment.

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Fig. 1. Representation of the formation of the two 3D models used in this work. In Model, where the MNPs are located just inside the cells. In&Out Model, where MNPs are located both inside and outside the cells. Results and discussion We have chosen spherical 11 nm iron oxide MNPs (11.2 ± 0.8 nm, Fig. 2), as this diameter is within the most commonly studied range for in vivo magnetic hyperthermia experiments.39 This diameter fulfils the requirements for in vivo experimentation, as it avoids fast renal clearance. This size is also considered to be in the range to achieve the better heating properties for magnetite/maghemite.40 MNPs have been prepared by thermal decomposition to have a careful control of the particle size and size distribution (Fig. 2). The superparamagnetic behaviour at room temperature, desired in many biomedical applications to prevent the MNP aggregation associated to a permanent magnetic moment,1 has been verified by magnetic measurements confirming the negligible coercivity and also indicating the good crystalline properties of the prepared material by a high saturation magnetization (Ms = 81 Am2/kg Fe3O4) (Fig. 1).

Fig. 2. Magnetic nanoparticle characterization. (A) TEM micrograph, (B) Particle size distribution analysis and (C) Field dependent magnetization of the MNPs in water. MNPs have been subsequently coated with a polymer (PMAO, poly (maleic anhydride-alt-1octadecene) modified with a fluorophore (TAMRA, Carboxytetramethylrhodamine). The polymer coating allows the transference of the magnetic cores from the organic to the aqueous phase and provides carboxylic groups that allow both the incorporation of the fluorophore, to track the location of the MNPs in the in vitro experiments, and glucose, to prevent the aggregation in cell culture medium and provide an active targeting molecule for the particles uptake.41 Glucose is especially interesting in our case as macrophages, as well as 5



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many tumour cells, due to their high energy requirements present upregulated glucose transporter proteins and subsequently the uptake of glucose-functionalized MNPs is enhanced.42 The successful glucose functionalization, routinely used in our lab,

43

has been

verified by ζ – potential measurements through a significant decrease of the MNP negative charge from -36 mV, before functionalization, to -13 mV after the glucose addition. Several tests have been performed to assure that the material was suitable for in vitro experiments. Stability of functionalized MNPs has been assessed by Dynamic Light Scattering (DLS) measurements, showing that the hydrodynamic size of the particles before and after the glucose functionalization remains below 70 nm for MNPs at pH = 7, suspended both in water and complete culture medium (with 10% Foetal Bovine Serum) (Table. S1, Supporting Information). In addition, a sterility assay was performed and no evidence of the presence of microbial colonies has been observed after performing the test with the MNPs (Fig. S1, Supporting Information). Finally, although the low toxicity of particles prepared following the same protocol has already been checked in the past,44-45 cell viability after the MNP administration was studied by flow cytometry in 2D cell culture models at different concentrations up to 100 μg of Fe per well (200 μg/ml), observing only a significant reduction of the cell viability at the highest concentration (Fig. S2, Supporting Information). From these results, an iron amount of 100 μg of Fe per well has been chosen for the hyperthermia experiments, as it is the highest concentration tested in which the viability is not significantly different to the controls. The low toxicity of this amount of MNPs has also been confirmed by flow cytometry in the 3D cell cultures (Fig. S3, Supporting Information). The heating efficiency of the MNPs in water suspensions has been evaluated before the in vitro test. The Specific Absorption Rate (SAR) of the MNPs measured at an iron concentration of 1 mg/mL and using a field amplitude of H = 20 kA/m, and a frequency of 829 kHz is 253 W/g Fe. Establishing a correlation between the heating capacity of the MNPs in suspension and their ability to heat efficiently in in vitro experiments is not straightforward. First, it is difficult to determine and mimic the aggregation degree of the MNPs in the cellular environment. It is also a complex task to know the local concentration of the MNPs once they have been internalized by the cells. However, until all these problems are solved, the most common approach to validate that a specific material produces heat in the presence of an AMF is to measure the change of temperature over time in water at physiological pH, as at least it provides some information for comparison with other reported values.

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Two different 3D cell culture models have been developed. To achieve the generation of two 3D cell culture models we have followed two different strategies for the incubation of the cells with magnetic nanoparticles. As a proof of concept, macrophages have been selected as a model cell line, as they are known to easily uptake this kind of particles.38, 46 The first approach followed has been to incubate the detached macrophages with the MNPs and, after removal of the particles that were not internalized, to form the 3D cell culture with the cells containing the MNPs. This approach leads to a 3D cell culture in which the MNPs are homogeneously distributed amongst all the cells and only located inside them (In Model), as observed by confocal microscopy and flow cytometry (Fig. 3), showing that almost all the cells (> 99%) contained MNPs after 1 h of incubation time. The second approach has been to embed the macrophages within the 3D structure and then administer the MNPs. This way, particles need more time to penetrate the collagen structure to reach the inner cells. Although, after the incubation time, the supernatant is removed and the 3D structure is washed, some MNPs remain within the collagen matrix outside the cells. Because in this model particles could be found located both inside and outside the cells, we have named it “In&Out Model”. The presence of particles both inside the cells and within the collagen matrix has been verified by confocal microscopy (Fig. 3). Interestingly, two different cell populations have been identified by flow cytometry: one population of cells with particles (58 ± 5%) and the other without particles (42 ± 8 %), indicating a slower rate of MNP uptake when compared to the In Model for the same incubation time of 1 h (Fig. 3), probably due to the time required for the movement of the MNPs towards the inner parts of the 3D structure. One of the advantages of using these two 3D cell culture models is that, although the same amount of particles has been administered to the cells, we have been able to achieve two different kinetic behaviours of MNP internalization.

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Fig. 3. 3D cell culture characterization (A, B, C, D) Confocal images. The nucleus is shown in blue (DAPI), actin in green (Phalloidin_AlexaFluor488) and MNPs in red (TAMRA). Scale bar: 10 µm. (E, F, G, H) Flow cytometry analysis of nanoparticle uptake. Data have been selected as a representation of a series of five experiments.

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AMF exposure enhances MNP uptake. Once it has been verified that the two models were successfully generated, the transformations that may occur with time have been evaluated. In particular, the capacity of the cells to continue internalizing MNPs in the In&Out Model has been assessed. To evaluate the time frame of the MNP uptake in the In&Out Model, the evolution with time of the two different cell populations previously observed by flow cytometry (with and without MNPs) has been measured (Table 1). Results have been compared also with 3D cell cultures of the In&Out Model after the AMF exposure to evaluate its impact on the MNP uptake. The percentage of cells that contain MNPs in the In&Out Model increases with time, reaching a point at 24h after their addition where all cells contain MNPs (Fig. 4). This uptake, however, is significantly faster in the 3D cell cultures exposed to the AMF. It has been observed that immediately after the AMF exposure, a higher percentage of cells had uptaken MNPs (70 ± 3 %) in comparison with the control experiment with no AMF exposure in which only 58 ± 4 % of the cells showed MNP internalization. In addition, 3 h after the administration of the MNPs, the two populations of cells (with (69 ± 4 %) and without (31 ± 4 %) particles) were still observed in the 3D cell culture that had no exposure to the AMF while almost all the cells exposed to the AMF (99 ± 0.5 %) had uptaken MNPs. Nevertheless, although all the cells from the In&Out Model contain MNPs 24 h after the 3D cell culture generation, still many particles remain located outside the cells within the collagen fibres as observed by confocal microscopy (Fig. 5). As a conclusion of these results, flow cytometry studies in the In&Out Model demonstrate an enhancement in the rate of the MNP uptake by the cells triggered by AMF exposure (Fig. 4). Experimental conditions -AMF +AMF

Time points 3h 24 h 69 ± 4 % 99.5 ± 0.3 % 99 ± 0.5 % 99.2 ± 0.6 %

0h 58 ± 4 % 70 ± 3 %

Table 1. Percentage of cells that contain MNPs in the In&Out Model at different time points, either in the control experiment with no AMF exposure (-AMF) or after the AMF exposure (+AMF). Data obtained by flow cytometry (n=5).

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Fig. 4. Flow cytometry analysis of the time dependent nanoparticles uptake in the In&Out Model. Left: Cells without exposure to the AMF (-AMF), Right: Cells after the AMF exposure(+AMF). (A, E) Control cells without MNP, (B, F) 0 hours, (C, G) 3 hours, (D, H) 24 hours. Data have been selected as a representation of a series of five experiments.

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Fig. 5. 3D reconstruction of confocal images acquired for the In&Out Model 24 h after AMF

exposure.

The

nucleus

is

shown

in

blue

(DAPI),

actin

in

green

(Phalloidin_AlexaFluor488) and MNPs in red (TAMRA). The presence of MNPs is still observed outside the cells 24 h after the AMF exposure. To further investigate the mechanisms behind this MNP internalization enhancement mediated by AMF exposure, two 3D cell cultures of the In&Out Model have been prepared, and one of them has been exposed to the AMF. Images of the whole 3D structure have been acquired for the initial time point (Fig. 6). In the case of the cell culture not exposed to the AMF, most of the particles have not been able to diffuse towards the inner part of the collagen matrix, being mainly localized in the outer areas of the 3D structure. However, in the cell culture exposed to the AMF, particles have penetrated better and are more homogeneously distributed throughout the whole 3D structure. This phenomenon of a more uniform distribution of MNPs after the AMF exposure has also been previously observed in a spheroid model of triple negative breast cancer.34 It has also been previously reported on a in vivo model where the nanoparticles where initially located in the collagen-rich outer areas of the tumour and penetrated more deeply into the core after the hyperthermia treatment.47 The enhancement of the MNP uptake may be associated with an increase in collagen permeability induced by the temperature rise during the MH treatment making the MNPs more “accessible” to the cells. It is known since a long time that the melting temperature of the triple helixes of the collagen type I, which is the most abundant in the nature, is few

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degrees above body temperature.48 Most recent studies have shown that the denaturation of the collagen vitrified gels takes place in two steps: a reversible process that results in a metastable collagen matrix, and an irreversible process that takes place at 60 ºC, leading a random uncoiled collagen.49 These results are relevant for the design of future preclinical treatments, as collagen is one of the major components of the tumour extracellular matrix present in breast,50 prostate,51 glioma,52 or pancreatic53 cancer. Therefore, the benefits of the application of periodical cycles of AMF exposure would be to improve the permeability of the extracellular matrix and therefore the access of chemotherapeutic drugs or cells from the immune system to the inner areas of the tumour. In addition, the enhancement of the internalization of MNPs after each cycle could positively influence subsequent hyperthermia cycles and this amplification effect could have positive synergetic effects in the final effectiveness of the treatment.

Fig. 6. Map image of confocal microscopy of 3D cell culture without (A) and with (B) the AMF exposure for the In&Out Model (0 hours). The image shows the overlay of two channels: green fluorescence of the labeled cells and red fluorescence of the MNPs. Scale bar is 500 µm. Magnetic Hyperthermia treatment induces different cell death mechanisms in the two 3D cell culture models. To evaluate the effect of the differences between the two models on the cell death mechanisms we have evaluated the activation of the cell death routes at three different time points (0, 24 and 48 h) after the hyperthermia treatment. Interestingly, the results have shown differences in the cell death mechanism between the two models. Just

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after the hyperthermia treatment (0 h) most of the cells remain alive in both models, being negative for Propidium Iodide (PI) and Annexin V staining. PI is a small molecule that enters in the cells and binds DNA only when the lipid bilayer membrane is disrupted, which happens in the late stages of the apoptosis and in the necrosis mechanism. Annexin-V binds phosphatidylserine (PS), a phospholipid that is normally located on the inner side of the cell membrane but translocates to the extracellular side in the first stage of the apoptosis. Therefore, cells that are positive for PI and negative for Annexin V have their cell membrane integrity altered without exposing the phosphatidylserine, a process typical from the beginning of necrosis, while cells that are positive for both markers indicate a late necrosis or late apoptosis stage.54 One day after the treatment, the In Model shows a significant population of necrotic cells (39.02 ± 4%) (positive for PI and negative for Annexin V), 60.09 ± 4.5% of cells that are still alive (negative for both markers) and a small percentage of cells (0.89 ± 0.1%) that are probably in a late necrosis stage (positive for both markers). On the contrary, at 24 h, the In&Out Model presents a very different situation where 38 ± 5.6% of the cells are undergoing apoptosis (positive for Annexin V and negative for PI) and the rest of the cells (61 ± 5.8%) are alive (Fig. 7). As a conclusion we can say that the In&Out Model results in a more controlled cell death pathway while the response to the MH treatment in the In Model is more aggressive.

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Fig. 7. Analysis of magnetic hyperthermia-induced cell death (Annexin V and PI staining). Selected density plot (representative of 5 experiments) obtained at 0h and 24h after MH treatment for the In Model (A) and In&Out Model (B). (C and D) Summarized flow cytometry data resulting from five independent experiments shown as mean ± SD. Statistical significance between the means at the different times was determined using a two-way 14


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ANOVA with Sidak's multiple comparisons test (**** p < 0.0001; *** p < 0.001; p > 0.05 no significance).

To evaluate the status of the cells that remain alive 24 h after the treatment, the cell cycle has been analysed by flow cytometry (Fig. 8). It has been previously shown that among the crucial events triggered by a heat shock stress are the changes in the cell cycle progression. Depending on the duration and the intensity of the heat shock, cells can enter into a transient or permanent cycle arrest that can take place either in the G1 or G2/M transition. Cells accumulate at these so-called checkpoints because the activity of the proteins that regulate these transitions (cyclin-dependent kinases) decreases.55 G1 checkpoint checks several aspects of the cells such as the size, nutrients and DNA damage before entering in the replication phase, and G2 checkpoint is focused on analysing DNA damage after the replication and replication completeness. So, depending when the DNA damage occurs the cells will be arrested in different phases. Previous results have shown that mild heat shock provokes a cell-cycle arrest in the G2/M phase.56 Cell cycle analysis of the control 3D cell culture has revealed that around 40% of cells are in the G2M phase in both models. This phase includes both cells undergoing the G2 phase, where cells prepare themself for mitosis, and also those cells that are dividing (M phase).57 The observation of an important percentage of cells in this sub-phase suggests a normal replication of the cells after the 3D model construction. In contrast, the analysis of the cell cycle from the cells that remained alive 24 h after the MH treatment for both models has revealed a significant decrease on the percentage of cells in the G2M and S phases, accompanied by an increase on the percentage of cells in the G0/G1 phase. These results indicate an arrest of the cells in the G0/G1 phase that does not allow the cells to duplicate its DNA and undergo the mitosis process. Therefore, it can be concluded that cells are suffering damages that are being detected by the key proteins preventing the cell replication process. The arrest is however more pronounced for the In Model. This result is probably a consequence of the heterogeneous distribution of MNPs along the 3D structure of the In&Out Model when the AMF is applied, as the core of the 3D cell culture would probably contain cells that have not incorporated MNPs or that are far away from other extracellular MNPs that act as heating agents. However, in order to have a more detailed explanation of the mechanisms involved, further experiments on the expression and activity of some proteins such as p-53 and some cyclin-dependent kinases should be performed in future studies. 15



It should also be briefly discussed that a small decrease in the percentage of cells in mitosis phase is observed in the In Model in comparison with the In&Out Model. This effect has been observed both in the model with cells incubated with MNPs and the control incubated without MNPs and is probably due to the protocol of the 3D model preparation. In the In Model, the cells have been maintained in suspension during 1 hour for the incubation period. As RAW–264.7 are adherent cells, the suspension step is less favourable for the normal cell development in comparison with the In&Out Model, where the cells are directly located into the collagen matrix.

Fig. 8. Effect on cell cycle distribution 24 h after magnetic hyperthermia treatment. (A) In Model (B) In&Out Model. The distribution of cells in each phase is given in the case of no MH treatment (I); after MH treatment (II) and the comparison of both experiments (III). Results are represented as average ± S.D. and are based on three independent experiments. Statistical significance was determined using a one-way ANOVA with Bonferroni post-test (***p

Dual role of magnetic nanoparticles as intracellular hotspots and

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